Isolation Of Deoxyribonucleic Acid Research Articles

Identification of differentially expressed genes controlling the expression of flowering in Bambara groundnut (Vigna subterranea [L.] Verdc.)

Bambara groundnut flowering is a crucial developmental stage in the vegetative to reproductive period. The earliness to lateness of flowering is regulated by various interconnected genetic pathways encoded by genes. Deoxyribonucleic acid (DNA) of the selected accessions was extracted through leaf samples at 3 weeks old, using Dellaporta Miniprep for Plant DNA Isolation procedure. The high-quality DNA was sequenced using Diversity Arrays Technology (DArT) markers and single nucleotide polymorphisms (SNP’s) associated with flowering was identified. There is need to investigate the genetic make-up of the cleistogamous flower of Bambara through associated genes for improvement. This research work identified four markers associated with the flowering of Vigna subterranea and the role of variant identified genes in flowering. The identified markers from the sequence and the selected amino acid sequence were used as a query to search the legume protein database in Vigna radiata. The four markers with adequate information associated with flowering in the sequence were 24385352|F|0–28:T > C-28:T > C; 27641816|F|0–17:C > T-17:C > T; 24384204|F|0–24:C > T-24:C > T and 24346601|F|0–67:T > C-67:T > C and significant at P < 1.68 × 10−4 at chromosomes 7, 11, 4, and 5. The identified genes including histones, Polyketide, cyclase/dehydrase, Transcription factor MYC/MYB N-terminal, Rhamnogalacturonate lyase, DHHC-type zinc finger protein, Putative S-adenosyl-L-methionine-dependent methyltransferase, Ribosomal protein L2, D-galactoside/L-rhamnose binding SUEL lectin domain, Lipase GDSL, Histone deacetylase superfamily, Basic-leucine zipper domain, TUP1-like enhancer of split, Zinc finger ZZ-type, Homeodomain-like, Phosphatidylethanolamine-binding protein PEBP, Leucine-rich repeat which are tools in controlling flowering in Bambara groundnut. This study revealed that Bambara groundnut flowering is controlled by the interplay of genes.

Novel 3D-Printed Microfluidic Magnetic Platform for Rapid DNA Isolation.

This study presents a novel miniaturized device as a 3D-printed microfluidic magnetic platform specifically designed to manipulate magnetic microparticles in a microfluidic chip for rapid deoxyribonucleic acid (DNA) isolation. The novel design enables the movement of the magnetic particles in the same or opposite directions with the flow or suspends them in continuous flow. A computational model was developed to assess the effectiveness of the magnetic manipulation of the particles. Superparamagnetic monodisperse silica particles synthesized in-house are utilized for the isolation of fish sperm DNA and human placenta DNA. It was demonstrated that the proposed platform can perform DNA isolation within 10 min with an isolation efficiency of 50% at optimum operating conditions.

Inventory and quality control of biosample collection from pregnant women at different gestational ages to search for early biomarkers of pregnancy complications

Aim. To conduct an inventory and quality control of biosample collection from pregnant women at different gestational ages to search for early biomarkers of pregnancy complications.Material and methods. In this work, methods for assessing the sample preparation of biosamples were used, including the isolation of deoxyribonucleic acid (DNA)/ribonucleic acid from various biomaterials, polyacrylamide gel electrophoresis of protein, and database analysis.Results. Inventory and quality control of the collection (n=18390) was carried out, which confirmed the high safety of the biomaterial, regardless of storage period. The mean concentration of DNA was 69,96±6,56 ng/µl, extracellular DNA (ecDNA) — 0,20±0,02 ng/µl, ribonucleic acid — 38,16±5,69 ng/µl. DNA Integrity Number (DIN) &gt;9, RNA integrity number (RIN) &gt;7, A260/280 &gt;1,8 were for all studied samples. Protein electrophoresis demonstrated no degradation of protein zones after longterm storage. The number of errors detected during the inventory was 84 (0,46% of all records in the database), while there were 64 donors with incomplete clinical information (15% of all donors in the collection).Conclusion. The necessity of mandatory implementation of standard operating procedures when creating and maintaining a collection, on the one hand, and periodic inventory with biosample quality assessment, on the other, has been demonstrated.

Vitamin D Receptor Gene Polymorphisms and Association with Vitiligo in Indonesian Population.

Vitiligo is an acquired depigmenting skin disorder due to the loss of melanocyte function in the epidermis and hair follicles. The pathogenesis of vitiligo is multifactorial, with genetics being a predisposing factor. Previous studies had varying results regarding whether or not polymorphisms of vitamin D receptor (VDR) gene are associated with the risk of vitiligo in specific populations. This study investigated the association between three frequently analyzed VDR gene polymorphisms (ApaI, BsmI, TaqI) and susceptibility to vitiligo in Indonesian population. Thirty-four vitiligo patients and 34 age- and sex-matched healthy subjects aged ≥18 years old were recruited in the Dermatology and Venereology Outpatient Clinic of Dr. Hasan Sadikin General Hospital, Bandung, Indonesia. Genomic deoxyribonucleic acid (DNA) was extracted from the peripheral blood using a DNA isolation kit. VDR gene polymorphisms (ApaI, BsmI, and TaqI) were investigated using the polymerase chain reaction-restriction-fragment length polymorphism method. The differences of genotype distributions and allele frequencies were statistically compared between case and control groups using Chi-square test. VDR gene polymorphisms were identified in 68 participants, consisting of Aa (n = 14), aa (n = 20), Bb (n = 15), bb (n = 19), and TT (n = 34) genotypes in the case group. In the control group, Aa (n = 6), aa (n = 28), Bb (n = 17), bb (n = 17), and TT (n = 34) genotypes were identified. However, only subjects with ApaI Aa genotype polymorphism had a 3.267-fold increased risk of developing vitiligo. This study showed that ApaI Aa genotype polymorphism of the VDR gene increases the risk of vitiligo in Indonesian population.

Исследование методов экстракции ДНК из сырого молока

This article presents the results of work on the study of the effectiveness of various methods of DNA isolation for use in the analysis of biosafety of milk and dairy products by PCR. Some components of milk, such as proteases and calcium ions, can significantly reduce the effectiveness of PCR and lead to distortion of the results. We have given quantitative (DNA concentration) and qualitative assessments (amplification followed by gel electrophoresis) to commercial DNA purification kits «DNA-sorb-S-M» and «DNA-Extran-2». The results obtained indicate the applicability of the selected kits for the isolation of deoxyribonucleic acids from raw milk, the quality and quantity of which allows them to be used for the successful subsequent molecular genetic analysis of the safety and authenticity of milk.

A New Proposed Symbiotic Plant-Herbivore Relationship between Burkea africana Trees, Cirina forda Caterpillars and Their Associated Fungi Pleurostomophora richardsiae and Aspergillus nomius.

Burkea africana is a tree found in savannah and woodland in southern Africa, as well as northwards into tropical African regions as far as Nigeria and Ethiopia. It is used as fuel wood, medicinally to treat various conditions, such as toothache, headache, migraine, pain, inflammation, and sexually transmitted diseases, such as gonorrhoea, but also an ornamental tree. The current study investigated the possible symbiotic relationship between B. africana trees and the C. forda caterpillars and the mutual role played in ensuring the survival of B. africana trees/seedlings in harsh natural conditions and low-nutrient soils. Deoxyribonucleic acid isolation and sequencing results revealed that the fungal species Pleurostomophora richardsiae was highly predominant in the leaves of B. africana trees and present in the caterpillars. The second most prominent fungal species in the caterpillars was Aspergillus nomius. The latter is known to be related to a Penicillium sp. which was found to be highly prevalent in the soil where B. africana trees grow and is suggested to play a role in enhancing the effective growth of B. africana trees in their natural habitat. To support this, a phylogenetic analysis was conducted, and a tree was constructed, which shows a high percentage similarity between Aspergillus and Penicillium sp. The findings of the study revealed that B. africana trees not only serve as a source of feed for the C. forda caterpillar but benefit from C. forda caterpillars which, after dropping onto the soil, is proposed to inoculate the soil surrounding the trees with the fungus A. nomius which suggests a symbiotic and/or synergistic relationship between B. africana trees and C. forda caterpillars.

Assessment of extracted DNA from whole blood and buffy coat in healthy individuals

Deoxyribonucleic acids (DNA) are the genetic material in all organisms. Extraction of DNA is the initial gait for the field of molecular biotechnology and biomedical research to advance understanding of human illnesses from various diseases and provide practical solutions for diagnosis and treatment. Routine diagnostic techniques in medicine have been greatly enhanced by molecular biology. Due to research on deoxyribose nucleic acid, it frequently finds use in medicine. Efficient isolation of DNA from a sample is the basis for successful forensic DNA profiling. DNA can be extracted from variety of samples such as a whole blood, buffy coat, hair, skin, tissue, urine, buccal swab, saliva etc. Numerous molecular biologic applications require genomic DNA (gDNA) in high quality and in adequate quantity. We extracted genomic DNA from buffy coat and whole blood samples and compared the results of the product obtained in terms of quantity (concentration of DNA extracted and DNA obtained per ml of blood used) and quality (260nm/280nm ratio) of the obtained yield. The aim of this study was to extract DNA from whole blood and buffy coat and compare quantity or Concentration (μg/ml) and purity (260nm/280nm) of extracted DNA. The present study was a hospital- based comparative and experimental study. The study was conducted over a period of 1 year on 50 samples. The venous blood sample is taken from the healthy individuals who are willing to donate blood in blood bank. The 5 ml of blood sample taken by venipuncture from healthy individuals were equally dividing into two anticoagulant EDTA tubes by equal volume. The first tube was use as it is, as a whole blood sample and second tube were centrifuged to obtained three phase separation and prepare buffy coat. From both of the samples extraction of DNA is carried out by follow the protocols of modified salting out method. The integrity and purity of the extracted DNA are assessed by Thermo Scientific Nano Drop 2000Spectrophotometers. Our results show thatMean±SD of concentration of DNA from whole blood and buffy coat was 37.49±25.31 μg/ml and 101.83±31.22 μg/ml (P &amp;#60; 0.0001). The Mean±SD of purity (260nm/280nm) of DNA from whole blood and buffy coat was 1.66±0.16 and 1.74±0.15 (p≤0.05). The both p value was found to be statistically significant.The present study concluded that DNA extracted from Buffy coat had purity as well as mean concentration significantly higher when compared to DNA extracted from whole blood.

The Potency of the BMP 15 Gene as a Marker of Reproductive Traits in Sumba Ongole (Bos indicus) Cattle

Background: As one of the important genetic resources for Ongole beef cattle, the study aimed to identify any potential polymorphisms in the Bone Morphogenetic Protein 15 gene in a group of Sumba Ongole (SO) cattle. Methods: Blood samples from 161 SO cattle were obtained and examined. The initial stage was the isolation of deoxyribonucleic acid (DNA). The next procedures were electrophoresis and extraction, followed by annealing temperature optimization, amplification and sequencing. The Basic Local Alignment Search Tool (BLAST) was used to examine the sequencing findings. The annealing temperature was 58°C for 15 seconds, with an amplification gain of 350 base pairs (bp). Result: The result showed that there were six single nucleotide polymorphisms (SNPs) of the BMP 15 gene in this region. Six mutations were SNP c.57 G greater than A, SNP c.120 C greater than T, SNP c.338 G greater than T, SNP c.360 C greater than A, SNP c.361 G greater than T, SNP c.365 A greater than T. The polymorphic informative content (PIC) value (0.25 less than =PIC less than =0.5) was reached from low (0.02) to moderate high (0.45 and 0.47). A total of 2 SNPs in the BMP 15 gene with moderate-high PIC value was c.360 C greater than A and c.365 A greater than T. In conclusion, we reported for the first time the polymorphism of the BMP 15 gene which is required for further investigation as to whether this polymorphic could be responsible for reproductive trait variations in Ongole cattle.

Genetic diversity and population structure of some Nigerian accessions of Bambara groundnut (Vigna subterranea (L.) Verdc.,) using DArT SNP markers

Bambara groundnut is one of the crops with inadequate molecular research to show its full potentials. Previous studies showed morphological diversity with inadequate information to confirm genetic variations. In the quest to reveal the genetic potentials, deoxyribonucleic acid (DNA) of the selected accessions was extracted through leaf samples at 3 weeks old, using Dellaporta Miniprep for Plant DNA Isolation procedure. The high quality DNA was sequenced using Diversity Arrays Technology (DArT) markers to unlock diversity among Bambara groundnut of Nigerian origin. Cluster analysis (neighbor-joining clustering) of the single nucleotide polymorphisms (SNP’s) were used to generate sub-population to show relatedness and differences. Seven sub-populations were generated with 5927 (50.13%) high quality DArT markers out of the 11,821 SNPs generated. This revealed high genetic diversity existed among the selected Bambara groundnut accessions in Nigeria. This also revealed that DArT markers were highly efficient in classifying the accessions based on molecular expressions. This study also identified markers responsible for genetic variation that could facilitate the characterization of larger collections for further utilization of genetic resources and most importantly Bambara groundnut for the purpose of crop improvement.

Population features of alleles and genotypes frequency distribution of polymorphic genetic markers of antipsychotic medications pharmacokinetics in the Kazakh population.

The presented article is relevant, as the main goals of schizophrenia treatment are to achieve a response to psychopharmacotherapy, reduction and stabilization of psychopathological symptoms, qualitative remission, which in general implies the creation of a stable quality of life for the patient. The purpose of the study was to evaluate the population features of the frequency distribution of alleles and genotypes of polymorphic genetic variants of according to genome-wide association studies analysis of pharmacokinetics-associated antipsychotic medications, in an ethnically homogeneous Kazakh population. The research material was deoxyribonucleic acid (DNA) isolated from the peripheral blood of 1,800 conditionally healthy persons of Kazakh nationality. DNA isolation was carried out by the magnetic polyvinyl alcohol magnetic particle separation method. The analysis of the frequency distribution of the studied genotypes in the Kazakh population showed their compliance with the Hardy-Weinberg equilibrium for all studied polymorphisms (p > .05). The obtained results showed that CYP2C19 (rs4244285, rs4986893) polymorphisms occurs in Kazakhs significantly more often than European and a number of Asian populations, which significantly affects the decrease in effectiveness and increases the risk of side complications during therapy with antipsychotic medications in the Kazakh population.

Investigation of species distribution of nontuberculosis mycobacteria isolated from sputum samples in patients with suspected pulmonary tuberculosis.

Rapid and accurate identification of mycobacteria is important for the species-specific treatment of the disease. The aim of this study was the identification at the species level of 34 nontuberculous mycobacteria strains isolated from respiratory tract samples and 14 reference strains as by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Isolates derived from clinical specimens were subcultured in the Lowenstein-Jensen medium. Deoxyribonucleic acid isolation was carried out using the boiling method. PCR amplification was performed using primers specific to the hsp65 gene region. The PCR products were digested BstEII and HaEIII enzymes. All samples were studied comparatively by two different centers. In our study, the most common species were found to be Mycobacterium intracellulare in 23.52% (8/34). The performance of the PCR-RFLP method in detecting mycobacteria was found to be 82.35%. The PCR-RFLP method is a rapid, cheap, and practical method for the identification of mycobacteria.

Association of human papillomavirus in penile cancer: A single-center analysis.

ABSTRACTIntroduction:Human papillomavirus (HPV) is a known risk factor of penile cancer (PeCa). However, studies evaluating its true association are limited. In this study, we aimed to estimate HPV prevalence and its true association with PeCa in terms of molecular biological activities.Materials and Methods:This single-institutional prospective observational study was conducted between June 2016 and August 2019. We included 40 men with PeCa as a study group and 20 age-matched uncircumcised men who underwent circumcision for phimosis as a control group. Both the groups underwent deoxyribonucleic acid isolation for HPV subtyping followed by evaluation of relative E6/E7 messenger ribonucleic acid (mRNA) expression profile and relative telomerase activity in tissue samples. HPV-16 and -18 were categorized as high-risk, whereas HPV-6 and -11 were categorized as low-risk subtypes.Results:The mean (±standard deviation) age of PeCa was 51 ± 15.9 years. The majority of patients had stage II disease, and the most common procedure done was partial penectomy. The overall prevalence of HPV in PeCa was 42.5% (n = 17) as compared to 20% (n = 4) in controls. Among the subtypes, the most common subtype was HPV-16 noted in 33.3% (8/24) of cases, followed by HPV-18 in 29.2% (7/24) of cases. PeCa tissues had a significantly higher relative E7 mRNA expression for HPV-18 than the control group (P = 0.016). The mean relative telomerase activity was significantly higher in the PeCa tissues than the control group (138.66 vs. 14.46, P < 0.001). A significantly higher relative telomerase activity was noted in the PeCa tissues positive for high-risk HPV subtypes than controls (141.90 vs. 14.46, P = 0.0008), but not between high-risk HPV-positive and HPV-negative PeCa cases (141.90 vs. 137.03, P = 0.79). High-risk subtypes were not associated with tumor stage (P = 0.76) or lymph node metastasis (P = 0.816).Conclusions:HPV was associated in 42.5% of PeCa cases based on our experience from a single institution. PeCa tissues had a higher relative E7 mRNA expression for HPV-18 and relative telomerase activity as compared to controls suggesting their potential role as surrogate markers of virus-induced tumorigenesis.

Comparison of methods to extract PCR-amplifiable DNA from fruit, herbal and black teas

The success of polymerase chain reaction (PCR) assay depends on template deoxyribonucleic acid (DNA) being sufficient with respect to both quantity and quality. Some biological materials contain compounds which inhibit the functioning of DNA polymerase and thus need to be removed as part of the DNA extraction procedure. The aim of the present experiments was to optimise the process of DNA isolation from various types of black, fruit and herbal teas. A comparison was made between two cetyltrimethylammonium bromide (CTAB)-based protocols and two commercially available DNA purification kits. The yield and integrity of the extracted DNA were monitored both spectrophotometrically and using agarose gel electrophoresis. The presence/absence of inhibitors in the DNA preparations was checked by running quantitative real-time PCRs. The optimal protocol was deemed to be the CTAB method described in ISO 21571:2005, so this method is recommended for the routine sample analysis of tea products.

Study of myocillin gene variants alleles in primary open angle glaucoma patients and their first degree relatives in North West Rajasthan, India

Glaucoma is defined as progressive optic neuropathy leading to irreversible blindness if not treated on time. Primary open angle glaucoma (POAG) is most common form of glaucoma. Mutations in myocilin gene (MYOC) account for 2–4% of POAG cases. To identify and evaluate MYOC variants alleles among patients with POAG and their healthy first degree relatives. 66 POAG patients and 26 healthy first degree relatives recruited for study. All patients underwent complete ophthalmic examination followed by genomic DNA (deoxyribonucleic acid) isolation from peripheral blood and quantification of DNA on spectrophotometer. All samples were amplified with each primer by PCR (Polymerase Chain Reaction) technique and amplified DNA and primer sequence checked again by electrophoresis for confirmation of specified MYOC gene mutation. We identified a known MYOC missense mutation, Pro370leu in 16 POAG cases and found consistent genotypic but not phenotypic correlation in 4 of their first degree relatives. Out of 16 cases, pathogenic MYOC gene variant was found in 12 adult onset POAG, 3 juvenile onset POAG, and 1 case of OHT. This study is first of its kind in North India. Our study showed frequency of MYOC gene mutation in POAG cases was 24.24% which is much higher than found elsewhere in India and other countries (2-5%). Frequency of transmission of pathogenic MYOC gene variant in first degree relatives was 25%. The future outcome of our study is promising since early diagnosis and management of high risk family members is possible.

Presence of Specific Periodontal Pathogens in Prostate Gland Diagnosed With Chronic Inflammation and Adenocarcinoma.

BackgroundIntraprostatic inflammation is frequently observed in the prostate and linked to prostatic diseases, including prostatitis, benign prostatic hyperplasia (BPH), and cancer. The etiology of prostate diseases is unclear. Periodontal diseases are associated with an increased risk of prostate diseases. In men, chronic prostatitis and moderate/severe periodontitis have significantly elevated serum prostate-specific antigen (PSA) levels. Treatment of periodontal disease reduced PSA levels in men. The presence of periodontal pathogens deoxyribonucleic acid (DNA) was identified in the prostate fluid of prostatitis patients. These pathogenic bacteria might have the potential to trigger prostatitis progressing to prostatic adenocarcinoma. The mechanism(s) explaining the etiology of association between periodontal disease and prostate cancer remains unclear. However, the presence of periodontal pathogens has not been analyzed in the prostate gland.ObjectiveTo identify and compare the presence of specific periodontal pathogens in the areas of BPH, inflammation, and cancer of the prostate glands diagnosed with malignancy.Materials and methodsWhole-mount radical prostatectomy sections from men (n=30) were identified for BPH, inflammation, and cancer areas and marked for tissue procurement. The tissues were subjected to DNA isolation and analysis of microbial DNA and total bacterial load for the following pathogens, including Porphyromonas gingivalis strain ATCC 33277, Prevotella intermedia strain B422, Treponema denticola strain 35405, Fusobacterium nucleatum subsp. fusiform strain, Tannerella forsythia strain ATCC 43037, and Campylobacter​​​​​​​ rectus strain ATCC 33238performed real-time PCR. The universal bacterial primer pairs were used to detect genomic DNA (gDNA) from the total bacteria present in the samples. All species-specific primers were designed to target the variable regions of the 16S ribosomal RNA (rRNA). Data were analyzed using the 2-ΔΔCT method, statistically validated using unpaired t-test and ANOVA test.ResultsA total of 90 samples of prostate tissue specimens were analyzed for periodontal pathogens; only one pathogen (F. nucleatum subsp. fusiform strain ATCC 51190) showed a significant difference compared to the expression of S. epidermidis (internal control). In particular, F. nucleatum expression was 9, 11.9, and 10.3-fold higher in BPH, inflammation, and cancer, respectively, at p-value <0.05. Moreover, the bacterial load abundance/expression was almost similar in BPH (46.8-fold), inflammation (40.9 fold), and cancer (41.5 fold) higher. There was no significant difference in bacterial load (folder change) among the three areas of BPH, inflammation, and cancer (p-valve>0.05). Similarly, there was no significant difference between F. nucleatum (folder change) among the three areas (p-valve>0.05).Conclusion Fusobacterium nucleatum is identified in the prostates that harbor cancer, chronic inflammation, and BPH.

DNA isolation success rates from dried and fresh wood samples of selected 20 tropical wood tree species for possible consideration in forensic forestry

The successful isolation of DNA (Deoxyribonucleic Acid) is essential for the investigation process of forestry molecular genetics. Samples used are usually retrieved either from soft or juvenile plant organs because of their excellent DNA source. However, in certain cases, aforesaid samples are hard to obtain, as for forensic purposes. Alternatively, woods possess potential as alternative source of DNA whose extraction method has been developed with varying degrees of success. However, to date, effectiveness on tropical wood grown in Indonesia has not been widely reported. Therefore, objective of this study was to compare the results of DNA isolation of various dried and fresh wood samples by using two isolation methods: Cetyl Trimethyl Ammonium Bromide (CTAB) and Qiagen DNeasy Plant Mini Kit (QDPMK). Extraction results were visualized in agarose gels and quantified using Nanophotometer NP80 Implen which were then amplified using two universal primers: ITS and rbcL for detecting DNA signals. Extraction results from dried wood indicated no visualization in the gel, while fresh wood samples showed thick smeared bands on both extraction methods. Quantity test results denoted higher concentration in CTAB-extracted samples compared to samples extracted using QDPMK, in both types of samples, even though both resulted in optical density ratios outside the range of purity (λ260/280: 1,8–2,0 and λ260/230: 2,0, respectively). Success rates of ITS and rbcL primary amplification in dried wood samples were quite low yet outputs from the two methods did not differ significantly. Meanwhile, outcome of ITS and rbcL amplification on fresh wood samples had a fairly high success.

OPTIMIZED METHOD FOR THE EXTRACTION OF DNA FROM Sargassum liebmannii

Macroalgae contains high concentrations of polysaccharides, polyphenols and secondary metabolites. Those compounds are factors that prevent the isolation of deoxyribonucleic acid (DNA) and therefore inhibit the polymerase chain reaction (PCR) that is the beginning for the application of any molecular marker. In the present study, the application of six extraction methods was documented; four of them conventional and two commercial kits. The highest efficiency in DNA extraction was obtained with a conventional method with modifications. Said modifications consisted of immersing the algal tissue in ß-mercantoethanol and the addition of the DIECA salt in the extraction buffer. To test the purity of the DNA, in addition to the electrophoresis and spectrophotometry methods, a PCR was performed for the ISSR molecular marker, obtaining amplified fragments using the aforementioned modification.

English

Girardinia diversifolia (Link) Friis is a perennial herb commonly known as Himalayan giant nettle which belongs to the family Urticaceae. The plant has cultural, medicinal and economic importance. The plant contains high concentration of polysaccharides, polyphenols and secondary metabolites which obstructs the process of isolation of the Deoxyribonucleic Acid (DNA) and inhibits downstream Polymerase Chain Reaction (PCR) amplifications. This study protocol developed a DNA extraction protocol from leaf-tissue based on the Cetyltrimethylammonium bromide, and optimized the PCR protocol for Inter Simple Sequence Repeat (ISSR) analysis. Genomic DNA extraction process was conducted using modified Doyle and Doyle method to obtain good quality DNA. The method yielded 445 ng/µL of DNA, where the purity ranged from 1.8-2.0 indicating minimum contamination of metabolites. The optimum condition for ISSR analysis was established using 4 mM MgCl2 , 0.6 mM dNTPs, 2.0 U Taq polymerase, 50 ng template DNA, and 0.7 µM primer. PCR program was optimized in the sequence of denaturation at 94°C for 3 min, subsequently followed by 45 cycles at 94°C for 30 s, annealing temperature at 45°C for 30 s, extension at 72°C for 2 min, and final extension at 72°C for 10 min. The modified technique was found to be ideal for isolation of genomic DNA and optimization of PCR process for ISSR analysis of G. diversifolia. The results of the research are beneficial for future molecular characterization and genetic diversity analysis of allied taxa. Key words: Girardinia diversifolia, Himalayan nettle, DNA isolation.

Polymeric microcarrier for diagnostic assays and gene delivery systems

The interaction of deoxyribonucleic acid (DNA) with polymeric nano- and microcarriers has attracted significant attention in the studies on DNA diagnostic assays, DNA isolation and particularly gene delivery and cancer therapy agents. Therefore, in this study, plain poly(glycerol dimethacrylate) (poly(GDMA)) and poly(dimethylaminoethyl methacrylate)-grafted poly(GDMA) microgel particles were synthesized for high single-stranded DNA (ssDNA) adsorption and desorption capacity. Temperature- and pH-sensitive uniform poly(GDMA) particles were synthesized through a two-stage procedure. A GDMA dispersion polymerization procedure was used in the first stage of the study to obtain microgel particles with approximately 900 nm size. The surface-initiated atom transfer radical polymerization procedure, a living-polymerization technique of 2-dimethylaminoethyl methacrylate (DMAEM), was used in the second stage of the study to obtain polycationic molecular brushes on microgel particles. The resulting temperature- and pH-responsive microgels were used as a sorbent for ssDNA adsorption. Compared with commonly used sorbents, reasonably high ssDNA adsorptions were achieved with the proposed sorbent. The adsorbed ssDNA was desorbed from the sorbent at a high yield. The adsorption and desorption behaviors make poly(DMAEM)-grafted poly(GDMA) microgel particles a promising carrier/sorbent for ssDNA.

Molecular epidemiological study of multidrug-resistant tuberculosis isolated from sputum samples in Eastern Cape, South Africa

Drug-resistant tuberculosis prevalence is still a global challenge. Making it imperative to examine the molecular epidemiology of drug resistant tuberculosis. Molecular epidemiology methods can evaluate transmission patterns and risk factors, ascertain transmission cases of multidrug-resistant tuberculosis (MDR-TB) and furthermore determine transmission patterns in a human populace. This work focuses on MDR-TB isolates in distinguishing them into several species and genotyping the MDR-TB isolates, mainly for epidemiological studies using the genomic regions of difference and the spoligotyping techniques. A total of 184 deoxyribonucleic acid isolated from sputum samples that showed resistance against the two major first-line anti-tuberculosis drugs (Rifampicin and Isoniazid) were examined. The deoxyribonucleic acid samples were amplified with primers specific for each flanking region of the genomic regions of difference for the identification of different MTBC species. Isolates were further characterized into different lineages using the spoligotyping commercial kit. The M. tuberculosis species was detected in 83.7% (154/184) of the deoxyribonucleic acid isolates, followed by the M. caprae in 8.7% (16/184) and the least detected species was the M. africanum in 2.2% (4/184). Nineteen spoligotype international types (SITs) were identified in this study. The pre-existing shared types were from 94.6% (174/184) isolates with 1.1% (2/184) isolates recognized as orphans and 4.3% (8/184) isolates were not found in the SITVIT database. The predominant family (spoligotype) was the Beijing with 67.4% (124/184) strains. This study gives a general overview of drug resistant strains and the circulating strains in the Eastern Cape, South Africa and it shows that the common Mycobacteria in the province is the Beijing strain.